Production and characterization of monoclonal antibodies to Xenopus Proteins

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PROJECT SUMMARY: The amphibian embryo and in particular the frog Xenopus laevis is an outstanding model used by embryologist and cell biologist worldwide. This model system has led the discovery of axis formation (antero/posterior and dorso/ventral), tissue induction and nuclear reprograming among others. Advances in genome sequencing, proteomics and gene silencing have rejuvenated this model. In its most recent White Paper, the Xenopus community has identified a lack of adequate resource of antibody to study Xenopus proteins and proposed the production of such resources a priority. We propose to produce at least a hundred fully characterized monoclonal antibodies to transcription factors, cell surface proteins including signaling receptors and ligands as well as tissue specific markers. To achieve this goal we will immunize mice with both selected recombinant proteins expressed in mammalian cells and complex protein extracts isolated from Xenopus embryos. The recombinant protein targets will be selected by a steering committee composed of Xenopus investigators based on the importance of the proteins in developmental and diseases processes and the absence of cross-reactive commercial antibodies. From these immunizations we will produce and archive more than 10,000 clonal IgG-expressing hyrbidomas that will be screened by immunofluor- escence, whole mount immunostaining and immunoprecipitation to identify monoclonal anti-bodies to native proteins. Final characterization of monoclonal antibodies produced against the complex embryonic extracts will be performed by mass spectrometry followed by immuno-fluorescence on the recombinant putative target(s). These monoclonal antibodies, together with the characterization data will be freely distributed to the community via the developmental hybridoma study bank and Xenbase.